The present study was approved by the Ethics Committee of Zhejiang University (Hangzhou, China). All surgery and euthanasia of experimental mice was performed under sodium pentobarbital anesthesia to minimize suffering.

Hippocampal cells were isolated from 3×Tg-AD mice and cultured in 1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Hippocampal cells were treated with PI3K inhibitor LY294002 (2 mg/ml) and cultured in for 12 h a 37°C humidified atmosphere with 5% CO₂.

A total of twenty 6-week-old 3×Tg-AD mice (male, 28–35 g) with the Alzheimer's disease were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Shanghai, China). All animals were housed in a temperature-controlled facility at 23±1°C and relative humidity of 50±5% with a 12-h light/dark cycle with free access to food and water. The mice were divided into two group (n=10 in each group) and given intravenous injections of nicergoline (10 mg/kg body weight) or PBS (10 mg/kg body weight; both from Sigma-Aldrich; Merck KGaA) once a day. The treatment lasted 60 days.

Hippocampal tissues were isolated from the CA1 regions of experimental mice following a 60-day treatment with nicergoline (10 mg/kg body weight) or PBS. The brains were frozen in liquid nitrogen and following perfusion, fixation and cryoprotection, coronal sections were cut to 4 µm in a cryostat. After blocking with 5% Bull Serum Albumin (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C, free-floating sections were rinsed with PBS and placed in a solution with goat anti-mouse amyloid β (Aβ)-42 (1:1,000; cat. no. ab201060; Abcam, Cambridge, UK), Aβ-40 (1:1,000; cat. no. ab17295; Abcam) and amyloid precursor protein (APP; 1:1,000; cat. no. ab32136; Abcam) for 12 h at 4°C. Following incubation the sections were washed and incubated with a secondary rabbit anti-goat antibody conjugated with horseradish peroxidase (1:500; cat. no. A-11034; Chemicon International; Thermo Fisher Scientific, Inc.) for Aβ-42, Aβ-40 and APP staining for 1 h at 37°C. The sections were washed and observed using a fluorescent video microscope (BZ-9000; Keyence Corporation, Osaka, Japan) at magnification ×40. Immunohistochemical staining was used to examine the abundance of neuroprotection-associated proteins in the hippocampus. Immunohistochemical procedures were performed as previously described (29).

Hippocampal CA1 regions were isolated from experimental mice following treatment with nicergoline. Hippocampal neural cells were fixed in situ with bromodeoxyuridine and NeuN for 24 h at 4°C. The impairment of neurogenesis was analyzed under a fluorescence microscope (Olympus BX61; Olympus Corporation, Tokyo, Japan) at magnification 40×.

ATUNEL assay was performed to analyze apoptotic neurons in the hippocampus as previously described (30). Brain sections were viewed under alight microscope and of apoptotic morphology was labeled in sections. Magnification, ×40.

ELISA kits [Huiying Chemical Industry (Xiamen) Co., Ltd., Shanghai, China] were used to determine interleukin (IL)-1 (cat. no. MLB00C; Bio-Rad Laboratories, Inc., Hercules, CA, USA), tumor necrosis factor (TNF)-α (cat. no. MTA00B; Bio-Rad Laboratories, Inc.) and IL-6 (cat. no. M6000B; Bio-Rad Laboratories, Inc.) serum levels in mice. The procedures were performed according to the manufacturer's protocol. The final results were recorded at a wavelength of 450 nm.

The apoptosis of hippocampal neural cells was evaluated using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection kit (cat. no. ab14085; Abcam, Cambridge, UK) and a propidium iodide (PI) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Hippocampal neural cells were isolated from experimental mice and suspended with Annexin V-FITC and PI, according to the manufacturer's protocols. Fluorescence was measured via fluorescence-activated cell sorting using a flow cytometer (BD Biosciences). Cell apoptosis was analyzed using Expo32-ADC v. 1.2B software (Beckman Coulter, Inc., Brea, CA, USA).

Total RNA was extracted from experimental mouse hippocampal are as using RNA Easy Mini Extract kit (Sigma-Aldrich; Merck KGaA) and the RNA served as a template for cDNA synthesis by RT reaction using a PrimeScript RT Master mix kit (Takara Biotechnology Co., Ltd., Dalian, China). A total of one-tenth of the cDNA was used in the fluorescent RT-qPCR reaction using iQ SYBR-Green (Invitrogen; Thermo Fisher Scientific, Inc.). The PCR conditions included an initial denaturation step of 94°C for 2 min, followed by 30 cycles of 94°C for 30 sec, 59°C for 30 sec, 72°C for 2 min and a final elongation step at 72°C for 10 min. All forward and reverse primers were summarized in Table I. Relative mRNA expression was calculated by the 2^−ΔΔCq calculation (31). Results were presented as a fold difference relative to the housekeeping β-actin control gene.

Hippocampal cells were homogenized in radioimmunoprecipitation assay lysis buffer containing protease inhibitor (Sigma-Aldrich; Merck KGaA) and centrifuged at 6,000 × g at 4°C for 10 min. The supernatant was used for the analysis of target proteins using a protein extraction kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA), according to manufacturer's protocol. The protein concentration was measured in samples by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (10 µg) were separated on 12.5% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). SDS-PAGE was performed as previously described (32). For western blotting, primary antibodies: IL-1β (1:400; cat. no. ab200478), IL-6, TNF-α (1:500; cat. no. ab1793), Foxp2 (1:400; cat. no. ab16046), Src homology 2-containing inositol phosphatase (SxIP; 1:400; cat. no. ab45142), EB (1:400; cat. no. ab157217), P53 (1:400; cat. no. ab26), Bcl-2 (1:400; cat. no. ab32124), caspase-3 (1:500; cat. no. ab217), caspase-9 (1:500; cat. no. ab52298), Bid (1:400; cat. no. ab62469), Bax (1:400; cat. no. ab32124), Neprilysin (1:500; cat. no. ab79423), insulin (1:500; cat. no. ab32216), insulin-like growth factor-binding protein 3 (IGFBP-3; 1:500; cat. no. ab75988), vascular endothelial growth factor β (VEGF-β; 1:500; cat. no. ab53463), superoxide dismutase (SOD; 1:500; cat. no. ab13533), catalase (CAT; 1:500; cat. no. ab16731), glutathione (GSH; 1:500; cat. no. ab26255), PI3K (1:1,000; cat. no. ab182651; Thermo Fisher Scientific, Inc.), AKT (1:1,000; cat. no. ab8933; Thermo Fisher Scientific, Inc.), pAKT (1:1,000; cat. no. ab18260; Thermo Fisher Scientific, Inc.), ROS (1:1,000; cat. no. PA5-14732; Thermo Fisher Scientific, Inc.) and β-actin (1:2,000, cat. no. ab8226) were added following blocking with 5% skimmed milk for 1 h at 37°C, then the membranes incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies (1:5,000; PV-6001; OriGene Technologies, Inc., Rockville, MD, USA)for 1 h at 37°C. Finally, protein bands were visualized using WesternBright Enhanced Chemiluminescent substrate (Advansta, Inc., Menlo Park, CA, USA).

The cognitive competence of mice was determined by measuring the open field activity levels of mice in black plexiglass boxes (60×60×25 cm), to analyze the therapeutic effects of nicergoline. Mice were placed in open black boxes for 10 min and the behavior of the mice was monitored and evaluated using an auto-tracking system (EthoVision 8.0; Noldus, Wageningen, the Netherlands). A Morris water maze test was performed prior to and post-treatment with nicergoline to measure cognitive function. The Morris water maze experiment was performed three times in a circular stainless steel tank (155 cm diameter, 60 cm depth) filled with water to a depth of 40 cm (27.0±1.0°C) that was made opaque by the addition of skimmed milk. Mice learned to find a hidden circular platform (10 cm diameter, 1 cm below the surface of the water) in a fixed area in one quadrant of the tank.

Brain sections (30 µm thick) were prepared and stained with thionin for 2 h at room temperature. Images of stained tissues were digitally captured with an Olympus IX71 microscope controlled by DP manager software (Olympus Corporation, Tokyo, Japan) at a magnification of ×1.5.

All data are presented as the mean ± standard error of the mean. Statistical significance was determined using a two-tailed Student's t-test with SPSS 19.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance followed by Tukey's post hoc test, Kaplan-Meier analysis and log-rank statistical analysis were performed using GraphPad software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to analyze the efficacy of nicergolinein the treatment of Alzheimer's disease, mice with Alzheimer's disease received treatment with nicergoline (10 mg/kg body weight) or were administered PBS as a control. It was determined that nicergoline treatment markedly improved neurogenesis and cognitive competence compared with the control (Fig. 1A and B). In mice that received treatment with nicergoline, the degree of dementia was decreased following a 60-day course of treatment (Fig. 1C). Immunological staining indicated that pathogenic Aβ-42 and −40 peptides and APP were downregulated in the hippocampi of experimental mice (Fig. 1D). Levels of the neuroprotective forkhead box protein P2 (Foxp2), Src homology 2-containing inositol phosphatase (SxIP) and end-binding proteins (EB) were increased in hippocampi of nicergoline-treated mice (Fig. 1E). Additionally, marked differences in the dispersion of the pyramidal cell layer were observed between the nicergoline-treated and control groups, as determined by thionin staining (Fig. 1F). These results suggested that nicergoline exerted beneficial effects on neurogenesis and cognitive competence in mice with Alzheimer's disease.

As previously indicated, apoptosis of hippocampal cells is ubiquitous in patients with Alzheimer's disease (33). Therefore, the present study examined the apoptosis of hippocampal cells in a mouse model of Alzheimer's disease following treatment with nicergoline or PBS. It was demonstrated that nicergoline inhibited apoptosis in hippocampal cells isolated from mice with Alzheimer's disease and analyzed by flow cytometry in vitro (Fig. 2A). The TUNEL assay demonstrated that apoptotic hippocampal cells were significantly less prevalent in nicergoline-treated mice in vivo (Fig. 2B). In addition, the expression levels of apoptotic factors in hippocampal cells isolated from nicergoline-treated mice were investigated. The mRNA and protein levels of pro-apoptotic caspase-3, BCL2 associated X (Bax), BH3 interacting domain death agonist (Bid) and caspase-9 were downregulated in the hippocampal cells of nicergoline-treated mice (Fig. 2C and D). However, the mRNA and protein levels of anti-apoptotic p53 and apoptosis regulator Bcl-2 were upregulated in the hippocampal cells of nicergoline-treated mice (Fig. 2E and F). The results of the present study indicated that nicergoline may inhibit apoptosis in hippocampal cells by regulating the activation of apoptosis-associated genes.

The expression levels of inflammatory factors in hippocampal cells from experimental mice were examined. It was demonstrated that the plasma concentrations of IL-1, IL-6 and TNF-α were decreased in hippocampal cells treated with nicergoline (Fig. 3A-C). Neprilysin and insulin expression levels were upregulated in hippocampal cells in the mice with Alzheimer's disease treated with nicergoline for 60 days (Fig. 3D). In addition, insulin-like growth factor-binding protein 3 and vascular endothelial growth factor β protein levels were increased in hippocampal cells in the experimental mice treated with nicergoline (Fig. 3E). Oxidative stress differences between experimental mice we analyzed following treatment with nicergoline. As presented in Fig. 3F, the expression levels of reactive oxygen species, superoxide dismutase and glutathione were downregulated in hippocampal cells in the experimental mice treated with nicergoline. The results of the present study indicated that nicergoline mitigated Alzheimer's disease in mice via inhibition of inflammation and oxidative stress in hippocampal cells.

In order to elucidate the nicergoline-mediated mitigation of Alzheimer's disease in mice, molecular alterations in the PI3K/AKT signaling pathway in hippocampal cells were studied. As presented in Fig. 4A, treatment with nicergoline significantly increased the expression of PI3K and AKT in hippocampal cells isolated from experimental mice. In addition, nicergoline significantly promoted the phosphorylation of AKT compared with the negative control (Fig. 4B). It was observed that inhibition of PI3K activity (PI3KI) by LY294002 obviated the AKT expression and phosphorylation stimulated by treatment with nicergoline (Nicergoline + PI3KI; NiPI3KI) in hippocampal cells (Fig. 4C). In addition, the expression levels of IL-1, IL-6 and TNF-α were upregulated in PI3K-inhibitedhippocampal cells in vitro (Fig. 4D). Additionally, oxidative stress was increased in PI3K-inhibited hippocampal cells in vitro (Fig. 4E). Notably, the apoptosis rate was increased in PI3K inhibitor-treated hippocampal cells in vitro (Fig. 4F). The aforementioned results demonstrated that nicergoline regulated the activity of hippocampal cells through stimulation of the PI3K/AKT signaling pathway.